Project Summary Opioids are widely used to treat pain after trauma. Opioid use for pain management has dramatically in- creased, with little assessment of potential negative consequences for ongoing pain. Recent reports are critical of the lack of controlled, long-term studies to support the dramatic escalation of opioid treatment for chronic pain over the past decade. While one long-term concern is that there may be no benefit, another is that opioids could have negative consequences for pain. There would be major implications were opioid treatment to pro- long the course of pain long after opioid cessation. As described in this proposal, robust opioid-induced chroni- fication of pain does indeed occur, making this a phenomenon critical to understand. Disturbingly, we have discovered that opioids given around the time of trauma may be contraindicated: a brief course of treatment with morphine (5 mg/kg b.i.d. for 5-7 days) can amplify the magnitude and duration of neuropathic pain for months thereafter. Strikingly, this deleterious opioid effect occurs across all models tested to date: inflammatory pain, peripheral and central neuropathic pain, and post-operative pain, supportive that this is a widespread phenomenon worthy of study. This unanticipated effect of morphine across time and di- verse pain models had not been previously reported. Beyond our initial studies, nothing is known regard- ing the spinal mechanistic underpinnings of this multi-month exaggeration of neuropathic pain by a brief exposure to morphine restricted to the early post-trauma period. Three Aims are proposed. All studies are undertaken in both sexes, given that documented male/female differences in immune and glial function, neuropathic pain, and responses to opioids, suggest that distinct un- derlying mechanisms will likely be found across sexes. The first Aim examines how a short course of morphine in the early post-trauma period functionally modifies the neuroimmunology of the ipsilateral lumbar dorsal spi- nal cord and discovers which of these changes mediate pain enhancement. The second Aim utilizes state-of- the-art Robust Activity Marking (RAM) technologies in spinal cord to address how identified mediators of mor- phine-induced pain enhancement align with retrogradely labeled spinothalamic neurons with defined activation state. The third Aim examines supraspinal mechanisms contributing to morphine-induced chronification of neu- ropathic pain. Aim 3 utilizes state-of-the-art DREADD reversible inactivation of microglia vs. excitatory neurons to define the role of the caudal granular insular cortex (CGIC), which we have previously shown (in the ab- sence of early post-trauma morphine) to be critical to chronic pain maintenance. Here we will reversibly inhibit, in a cell-type targeted fashion, either microglia or excitatory neurons in CGIC either only during morphine dos- ing or only during the period of morphine-induced chronification of pain to define CGIC involvement in induction versus maintenance of this enhanced neuropathic pain state.